Fluorescent imaging is becoming an increasingly important technology for examining the real-time localizations of biologically relevant molecules within model organisms such as the zebrafish. Such imaging often requires the use of 2′,7′-dichlorofluorescein (DCF, FIG. 1), 2′,7′-difluorofluorescein (Oregon Green), or one of their derivatives since each offers pH insensitivity, compact size, and high quantum yield. However, the negatively charged carboxyl groups in these compounds can non-specifically interact with positively charged biomolecules. Furthermore, the carboxyl group is often found to be responsible when inherently cell-permeable molecules become impermeable following conjugation to fluorescein.
The Lindqvist group showed that the carboxyl group of these compounds was not essential for fluorescence emission. This observation was corroborated by the Nagano group at the University of Tokyo, who replaced fluorescein's carboxyl group with a methyl group, forming a compound, Tokyo Green, which demonstrated no loss of quantum yield. A similar methyl substitution was carried out by the Peterson group at The Pennsylvania State University, this time with Oregon Green, to produce Pennsylvania Green. In live human cells, Pennsylvania Green was more fluorescent than Tokyo Green due to its pH-insensitivity. While Pennsylvania Green is an excellent fluorescent probe, the synthesis of its conjugation-amenable derivatives is a lengthy process, requiring 10 linear steps. What is needed are fluorescent probes that are cell permeable and easily synthesized from commercially available materials.